Viscosity is an important factor to evaluate fluidity and diffusion of a condensed fluid, and is an important reference index of fluid diffusion rate as well. For a condensed fluid with macroscopically large volume, the methods and the devices such as rotational viscometer and falling ball viscometer for detecting fluid viscosity have been well developed, however these viscometers can only be used to detect fluid viscosity of macroscopically large volume, and at least 1 mL of fluid volume is needed. For viscosity detection of micro-environment at tissue level or cell level, these detection methods and devices generally can not be used. It is very meaningful to accurately detect viscosity of micro-environment at cell level, since viscosity is different in different position of a live cell, and cellular viscosity strongly influences diffusion and transportation of biomolecules and signals in cells. According to the work of K. Suhling and his coworkers, viscosity in membrane system micro-environment can reach up to 140 centipoise (cp) in a normal live cell, but viscosity in cytoplasm is merely 1-2 cp which is equivalent to that of water; intracellular viscosity is evidently increased when pathological changes happen in a cell and the cell becomes dead, and the highest viscosity can reach up to 300 cp, the obvious viscosity change in a cell would result in diseases or dysfunctions of organ and body. Therefore, it is necessary to develop a method to detect viscosity of micro-environment.
Recently, a method of utilizing a viscosity probe based on a fluorescent molecule rotor to detect viscosity of micro-environment in a cell was reported. The basic rationale for the viscosity probe involves fluorescence lifetime imaging and fluorescence ratiometry imaging. For the fluorescence lifetime imaging, much excellent work has been done. Emission wavelength of the fluorescence lifetime imaging only has a single emission peak, according to the work of A. Theodorakis and his coworkers, there are two methods for designing double-wavelength viscosity molecule rotor, one of which is a method based on photo-induced intramolecular charge transfer (ICT), but the probe designed according to the mechanism is easily influenced by solvent polarity; another method is to design molecule rotor based on fluorescence resonance energy transfer, but the method needs a good spectral overlap between emission spectra of energy donor and absorption spectra of energy receptor; furthermore, the various molecular orientations of the dipolar moments between the energy transfer pairs probably increase the uncertainties of final results, which inhibits the further design and application of the probe.
Precision and reliability are very important for cell imaging. Although Suhling etc. and Theodorakis's group developed fluorescence lifetime imaging and fluorescence ratiometry imaging to detect viscosity change, respectively. However, at present no fluorescence probe can be employed to evaluate viscosity by both methods at the same time.
Traditional pentamethine cyanine fluorescent dyes are synthesized by the reaction of a quaternary salt and a condensing reagent having a conjugated chain, with anhydrous sodium acetate as a catalyst in acetic anhydride solvent under argon atmosphere. Generally, pentamethine cyanine fluorescent dyes have advantages over the other fluorephores, e.g. large molar extinction coefficient (up to 105 level), near infrared absorption and emission and suitable fluorescence quantum yields etc., so they have been widely applied in protein labeling, DNA sequence analysis, ion and neutral small molecule recognition, and bioimaging in vitro and in vivo. However, up to now, it has not been reported that pentamethine cyanine dyes could be designed as molecular rotors sensitive to environment viscosity for detecting viscosity of solution or intracellular environment.